Achievements of the ES&T cover series of Qiu Rongliang's team of Sun Yat-sen University: Migration behavior and microscopic mechanism of environmental microorganisms in porous media

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First author: Jin Chao

Corresponding author: Qiu Rongliang

Communications: School of Environmental Science and Engineering, Sun Yat-sen University

Dissertation DOI:

https://doi.
org/10.
1021/acs.
est.
2c02899

https://doi.
org/10.
1021/acs.
est.
1c00785 (Cover Paper)

Picture summary

of the results of the introduction

Professor Qiu Rongliang's team from the School of Environmental Science and Engineering of Sun Yat-sen University has published two series
of articles on the migration behavior, microscopic mechanism and migration model of soil protozoa amoeba spores in porous media in environmental science & technology, a well-known academic journal in the field of environment 。 Through the investigation of the migration behavior of Dictyostelium discoideum spores in the pore medium, the anomalous migration behavior of its spore deposition efficiency decreases with the increase of ion strength (Environ.
Sci.
Technol.
, 55, 13, 8709-8720）； The host-endosymbiote relationship was revealed (D.
disc-based reticulum stalks).
Interaction between discoideum and Burkholderia agricolaris) on the physiological state of spores and their migration behavior (Environ.
Sci.
Technol.
, 2022, 56, 17, 12347-12357）
。 Through coupling column migration test, visualization of microfluidic devices, dissipative quartz crystal micro-celestial equality multi-scale research methods, and the first use of atomic force microscopy based on single-cell force spectroscopy technology, the microscopic stress between microbial spores and the surface of the environmental medium was determined, and the quantitative analysis of biocolloidal environmental behavior and its interface mechanism was realized.
Based on the independently developed nondestructive extraction technology of extracellular proteins, through the characterization of the physicochemical properties of the external spore membrane and the quantitative analysis of proteomics, the microscopic interface principle
of spores in response to changes in the external environment is proposed and verified to strengthen the agglomeration stability of extracellular proteins by regulating the spatial configuration and distribution characteristics of extracellular proteins, and then affect their mediated deposition process.
The migration behavior and microscopic mechanism of spores in the pore medium in the real environment are effectively analyzed, and the theoretical support
for the effective implementation of engineering measures and the management and control of environmental risks and ecological effects is provided.

introduction

Environmental microorganisms are ubiquitous in soil and groundwater media, frequent exchanges of matter and energy with the environment, and are the most active core of
biogeochemical cycle processes.
In recent years, the ravages of the COVID-19 pandemic and the widespread use of antibiotics and disinfectants have raised heights of concern
about the environmental behavior of microorganisms such as bacteria, spores and viruses.
Therefore, the analysis of the migration behavior and microscopic mechanism of environmental microorganisms in soil-groundwater is the key to understanding the transformation, transport and circulation of substances and elements in the critical zone of the earth, and is also the premise for
effectively assessing the risk of toxicity and pathogenesis of environmental microorganisms.
However, the heterogeneity of environmental microbial extracellular structures (e.
g.
, flagella, extracellular polymers, etc.
) and their complex life activities make it difficult to explain or predict their environmental behavior
using classical DLVO theory.
In response to this practical problem, we use the model amoeba (D.
As a research object, discoideum, the spores of Reticulum discoides are investigated by multi-scale research methods, so as to accurately evaluate their migration and deposition behavior in porous media, which provides a new method and theoretical perspective
for exploring the environmental behavior and regression of environmental microorganisms in soil and water.

Graphic guide

Scientific Discovery 1: Abnormal migration behavior and microscopic mechanism of environmental spores in pore media

（Environ.
Sci.
Technol.
, 55, 13, 8709-8720）

Firstly, the column migration experiment of spores was carried out, and it was found that as the ionic strength increased, the migration of spores in the effluent increased, and the interception in quartz sand decreased correspondingly (Figures 1a and b).

We ensured good dispersion of the spores in the suspension while ensuring consistent experimental conditions, and then further carried out mesoscopic scale deposition experiments
in QCM-D systems and visualization microfluidic devices.
The results showed the same trend (Figures 1c and d), which showed that the migration and deposition behavior of spores in porous media was significantly affected by ionic intensity, and their behavior was completely contrary
to theoretical predictions.

Figure 1.
(a) and (b) penetration curves (BTC) and interception curves (RP) of amoebic spores in quartz sand columns, respectively; (c) Typical deposition curve of amoebic spores in QCM-D; (d) Deposition images of spores in visualized microfluidic devices at 1, 10, 50 and 100 mM KCl ion intensities

Based on the results of series characterization of exospores, the mechanism of influence of exospores on the migration and deposition behavior of amoebic spores at different ionic intensities was investigated
.
The results showed that when the sporangia membrane was removed, the deposition rate of the spores decreased and the difference was significant at low ionic intensity, and the difference was small at high ionic intensity (Figures 2a and b).

After the statistics of the proportion of spores deposited by spore-spore interaction in the visualization microfluidic device, it was found that the sporin-spore interaction was significantly weakened after the removal of the sporozoic membrane (Figure 2c).

By analyzing the deposition membrane properties of spores in the QCM-D system, it was found that the deposition membrane structure of spores was relatively tight at low ionic intensity, that is, the interaction between spores and spores was strong
.
At high ionic strength, the reverse is true (Figure 2d
).

Figure 2.
The deposition rate of amoebic spores in (a) quartz sand columns and (b) visualization of microfluidic devices after removing the sporozoic membrane (_W0) (_W3); (c) Visualization of the proportion of spores deposited through spore-spore interactions in microfluidic devices; (d) Changes in the dissipation of spore deposition in QCM-D with frequency at 1, 10, 50 and 100 mM KCl ion intensities

In summary, the outer spore membrane of amoeba spores is significantly affected by ionic strength and dominates its migration and deposition behavior
.
Specifically, at low ionic intensity, the outer sporocyte membrane structure of the spores is relatively loose, and the spores-spores interaction is strong
.
Spores can enhance their retention
in a hollow medium by mediating deposition (i.
e.
, pre-staged spores promote the deposition of spores in the aqueous phase).
Under the high ionic strength, the outer spore membrane structure of the spores is dense, which greatly enhances
their migration capacity.
After removing the sporangium membrane, the difference in the deposition rate of amoebic spores under different ionic intensities became smaller, and the degree of influence of ionic strength on them was also reduced
.

Scientific Discovery 2: Microscopic mechanism of host-endosymbiote relationship influencing the environmental attribution of amoebic spores

（Environ.
Sci.
Technol.
, 2022, 56, 17, 12347-12357）

Spores in the environment will inevitably interact with bacteria, viruses and other microorganisms, which may significantly affect their environmental behavior and attributes
.
However, few studies have been studied
.
Therefore, we further investigate the mechanism
of the influence of the host-endosymbiote relationship on the migration and deposition behavior of amoebic spores in porous media under the influence of bacteria.
Column migration experiments, QCM-D and visualization of microfluidic device experiments have found that when interacting with bacteria, spore retention in porous media is enhanced
.

Figure 3.
At 1, 10, 50 and 100 mM KCl ion strengths, (a) D.
Penetration curve of discoideum spores in quartz sand columns (BTC); (b) Changes
in frequency resulting from spore deposition in the QCM-D system.

The foregoing work has shown that at low ionic strengths, D.
discoideum spores can enhance their deposition process
by mediating deposition.
Therefore, the pairs of bacteria carrying in the parallel plate chamber microfluidic system and the QCM-D system were compared.
Effects of
discoideum spore deposition.
The results show that the changes in the spores-spore surface proteins under different ionic intensity conditions promote the spores-spores interaction (Figure 6a), which also results in the deposition membrane of BCA spores in the QCM-D system being denser than that of FLA spores, and the spores are deposited on the quartz surface
by "holding clusters".

Figure 4.
(a) The proportion of BCA and FLA spores deposited through spore-spore interactions in parallel plate chamber microfluidic systems; (b) Changes
in the absolute values of ΔD/Δf deposited by BCA and FLA spores over time at the third overtone in the QCM-D system.

Proteomic assays
were performed on the outer sporomens of BCA (bacteria-containing amoeba) and FLA (free-living amoebae) spores using LC-MS/MS.
The results showed that the structure and properties of BCA spores surface protein changed significantly
.
The first is that its variety and number have been greatly improved (Figures 3a and B
).
Secondly, compared with FLA spores, the number of proteins on the surface of BCA spores on protein catabolic processes, protein hydrolysis, and cell adhesion functions increased significantly, while the number of proteins on the surface of FLA spores on cell differentiation (spores formation) increased (Figure 3c).

This shows that the surface protein of BCA spores is more conducive to its adhesion and reproduction process, while the protein on the surface of FLA spores is more conducive to its migration process
.

Figure 5.
(a) the amount of protein on the surface of the spores of BCA (bacteria-containing amoeba) and FLA (free-living amoebae); (b) The number of proteins up- and down-regulated in BCA and FLA spore surface proteins; (c) Functional annotations of BCA and FLA spore surface proteins (only the top 10 protein species with differential expression); (d) Isoelectric point distribution
of BCA and FLA sporin surface proteins.

In order to deeply explore the micromechanical mechanism of amoebic spore deposition on the quartz surface, the adhesion between BCA and FLA spores and the quartz surface was tested
by atomic force microscopy.
It was found that at low ionic strength, the adhesion between BCA spores and quartz surfaces was greater than that of FLA spores (Figure 4a
).
At high ionic strength, there is no significant difference (Figure 4b
).
This is because the electrostatic effect is weak at low ionic strength, and the protein on the surface of BCA spores plays a dominant role in cell adhesion function.
At high ionic strength, the electric double layer on the surface of the spores is greatly compressed, so that there is no significant difference
between the interaction between the two spores and the quartz surface.

Figure 6.
Under the conditions of 1 and 100 mM KCl ionic strength, (a) and (b) the maximum adhesion of BCA and FLA spores on the quartz surface and (c) and (d) the maximum adhesion force distribution and typical force curves

In summary, when interacting with bacteria, amoebic spores can develop and multiply
by directionally regulating the spatial structure and properties of their extracellular proteins to make it easier for them to adhere to the surface of the medium and then develop.
Therefore, its mediated deposition process is strengthened, so that its retention in porous medium is greatly improved
.

brief summary

Microbial spores are typical representatives of environmental microorganisms and are one of the most active parts of
the biogeochemical process in the soil and groundwater environment.
In this series of studies, we comprehensively evaluated the mechanism of ionic strength and outer sporozoic membrane on the migration and deposition behavior of amoeba spores in porous media by using multi-scale research methods, and further investigated the mechanism of action of the host-endosymbiote relationship (i.
e.
, interaction with bacteria) on the spores
.
It was the first to find that environmental microbial spores had abnormal migration behavior contrary to the expectations of classical DLVO theory in porous media, and the contribution mechanism
of heterogeneity of the surface of the outer spore membrane was analyzed.
The microscopic principle that amoebic spores change their migration efficiency by regulating the spatial configuration and distribution characteristics of extracellular proteins is proposed, which explains the internal reasons for
the penetration of spores into pore media in the real soil-groundwater environment.
At present, due to the complexity of the soil-groundwater environment and the difference in physical and chemical properties, it is often difficult to analyze the microscopic mechanism
of multi-interface and multi-process using traditional migration research methods 。 In the early stage of this study, a multi-scale interface visualization method for colloidal migration was constructed and improved, which can quantitatively evaluate the contribution of non-uniform properties in the interface of environmental microorganisms and media components from the perspective of molecular binding mechanism of extracellular structure, individual stress behavior at the single-cell level and response of microbial population, which provides an innovative means for clarifying the behavior and microscopic mechanism of environmental microorganisms, and provides a reference
for accurately assessing the environmental risks and ecological effects of toxic pathogenic microorganisms.

This project has been funded by the National Key R&D Program and the National Natural Science Foundation of China
.

 

About the AuthorFirst Author Jin Chao: Member of the Communist Party of China, Associate Professor of the Hundred Talents Program of the School of Environmental Science and Engineering of Sun Yat-sen University, doctoral supervisor, winner of the Young Scientist Award of the Chinese Society for Environmental Sciences (2022
).
He has long focused on the migration and transformation behavior and mechanism of colloidal pollutants in the soil-groundwater environment and the soil
remediation of mines and farmland.
He has presided over more than 10 domestic projects, including the National Key R&D Program and the National Natural Science Foundation of China
, and 6 foreign scientific research and industrial projects.
In Environ.
In important academic journals in the fields of Sci.
& Technol.
and Water Res.
, he has published 32 high-level papers as the first or corresponding author, authorized 2 national invention patents, 1 authorized utility model patent, and authorized software
copyright 。 He has won awards including the Guangdong Environmental Science Society Eco-environment Youth Science and Technology Gold Award (2022), the first prize of the Canadian Velocity Garage Entrepreneurship Competition (2019), the first prize of the AquaHacking Summit Entrepreneurship Competition (2017), and the nomination of the Canadian Doctoral Dissertation Governor Award (2015
).
As Executive Guest Editor, he organized the special issue of Water Magazine (2018-2020) and the special issue of Micromachines (2022-2023
).
Currently working as Environ.
Sci.
& Ecotechnol.
, Biochar, Chinese Chemical Letters and Bulletin of Environ.
Youth editorial board member
of magazines such as Contamination & Toxicology.
He was a member of the AWWA Coagulation and Filtration Committee of the American Water Association (2012-2015
).
Email: jinchao3@mail.
sysu.
edu.
cn
Corresponding author Qiu Rongliang: Vice President of South China Agricultural University, Professor and Doctoral Supervisor
of South China Agricultural University and Sun Yat-sen University.
Mainly engaged in heavy metal pollution soil plant-chemical-microbial joint remediation, water and soil environment organic pollution chemical remediation and bioremediation and other research
.
He has presided over more than 60 projects such as the National Key R&D Program, the National Natural Science Foundation of China, the National Natural Science Foundation of China Outstanding Youth Fund, the National 863 Key Project, the National Science and Technology Support Project, the Discipline Innovation Intelligence Introduction Base of the Ministry of Education, and the Innovation Team in Key Areas of the Ministry of Science and Technology, and published more than 330 research papers, of which more than 180 were included in SCI; Applied for 46 national invention patents, and 28 were authorized; He has been selected as the National Ten Thousand People Plan Science and Technology Innovation Leading Talents, the National Outstanding Youth Fund Winners, the Leaders of the Innovation Team in the Key Areas of "Soil Environmental Pollution Control and Ecological Remediation" of the Ministry of Science and Technology, the State Administration of Foreign Experts Affairs, the Ministry of Education's "Soil Environmental Pollution Control and Remediation" Discipline Innovation and Wisdom Introduction Base, the Ministry of Education's New Century Excellent Talents Support Program, the Guangdong Thousand Hundred and Ten Talents Project, the Guangdong Pearl River Scholars Distinguished Professor, the Guangdong Special Branch Program 100 Million Project Leading Talents, Outstanding teacher of Nanyue in Guangdong Province and recipient
of special government allowance of the State Council.
Email: eesqrl@mail.
sysu.
edu.
cn; qiurl@scau.
edu.
cn

Contributed by: Professor Qiu Rongliang's team
at Sun Yat-sen University.
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